Date of Award

August 2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Mark J McBride

Committee Members

Daad A Saffarini, Charles F Wimpee, Sonia L Bardy, Ching-Hong Yang

Keywords

Bacteroidetes, Carboxy Terminal Domain, Flavobacterium, Gliding Motility, SprB, Type IX Secretion System

Abstract

Flavobacterium johnsoniae exhibits rapid gliding motility over surfaces. At least twenty genes are involved in this process. Seven of these, gldK, gldL, gldM, gldN, sprA, sprE, and sprT encode proteins of the type IX protein secretion system (T9SS). The T9SS is required for surface localization of the motility adhesins SprB and RemA, and for secretion of the soluble chitinase ChiA. This thesis demonstrates that the gliding motility proteins GldA, GldB, GldD, GldF, GldH, GldI and GldJ are also essential for secretion. Cells with mutations in the genes encoding any of these seven proteins had normal levels of gldK mRNA but dramatically reduced levels of GldK protein, which may explain the secretion defects of the motility mutants. GldJ is necessary for stable accumulation of GldK and each mutant lacked GldJ protein. F. johnsoniae cells that produced truncated GldJ, lacking eight to thirteen amino acids from the C-terminus, accumulated GldK but were deficient in gliding motility. SprB was secreted by these cells but was not propelled along their surfaces. This C-terminal region of GldJ is thus required for gliding motility but not for secretion. The identification of mutants that are defective for motility but competent for secretion begins to untangle the F. johnsoniae gliding motility machinery from the T9SS.Proteins secreted by the T9SS typically have conserved C-terminal domains (CTDs) belonging to the type A CTD or type B CTD families. Attachment of 70 to 100 amino acid type A CTDs to a foreign protein allow its secretion. Type B CTDs are common but have received little attention. Secretion of the foreign protein sfGFP fused to regions spanning the SprB type B CTD (sfGFP-CTDSprB) was analyzed. CTDs of 218 amino acids or longer resulted in secretion of sfGFP whereas a 149 amino acid region did not. Some sfGFP was secreted in soluble form whereas the rest was attached on the cell surface. Surface-attached sfGFP was rapidly propelled along the cell, suggesting productive interaction with the motility machinery. This did not result in rapid cell movement, which apparently requires additional regions of SprB. Secretion of sfGFP-CTDSprB required coexpression with sprF, which lies downstream of sprB. SprF is similar in sequence to Porphyromonas gingivalis PorP. Most F. johnsoniae genes encoding proteins with type B CTDs lie immediately upstream of porP/sprF-like genes. sfGFP was fused to the type B CTD from one such protein (Fjoh_3952). This resulted in secretion of sfGFP only when it was coexpressed with its cognate PorP/SprF-like protein. These results highlight the need for extended regions of type B CTDs and for coexpression with the appropriate PorP/SprF-like protein for efficient secretion and cell-surface localization of cargo proteins.

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